Flow cytometers have been used extensively for analyzing cells and various particles. As a diagnostic tool, flow cytometers have been very effective in providing detailed information regarding the physiology of cells and particles. Flow cytometers have been used for analysis of cells and other particles in both research laboratories and in clinics. Flow cytometers include traditional hydrodynamically focused sample streams in a cuvette, a hydrodynamically focused sample stream with a jet-in-air sorting flow cytometer, and microfluidic systems that do not use focusing. For purposes of this application, the term “flow cytometer” should include all of these different types of flow cytometers.
In many typical flow cytometers, particles or cells that are of interest may be tagged with a marker, e.g., a fluorescing indicator, that may be stimulated to provide a quantifiable response, e.g., to emit light that may be detected by optical sensors. For example, in a typical flow cytometry system, a sample may be mixed with a fluorescent indicator that is known to bind to particles of interest and, in doing so, become photoreactive to a particular wavelength or wavelengths of light. The sample may then be focused into a stream or other constrained area and illuminated with high-intensity light of that particular wavelength or wavelengths—any photo-reactive indicator that is present will then fluoresce in response to such illumination. Such indicators normally are selected to emit light of other wavelengths than the stimulating light. Light that is emitted from the stimulated indicator may then be captured and measured to provide an estimate of how much indicator was present and fluorescing, thereby allowing for quantification of the amount of particles to which the indicator is bound.
In practice, there are many hurdles to obtaining such a measurement. For example, the intensity of stimulating light that must be provided to the indicator in order to cause it to fluoresce at a detectable intensity level at the desired wavelength may be several orders of magnitude higher than the intensity with which the fluorescing light is emitted. Since the target cells and particles are typically quite small in size, the stimulating light may need to be tightly focused on the cells or particles in order to provide sufficient stimulating light intensity without needlessly increasing the energy expenditure needed to stimulate the indicator.
Furthermore, a fluorescing particle or cell may emit fluorescing light in a generally omnidirectional manner, thereby making it impractical to efficiently capture all of the light that is emitted via fluorescence. This reduces the amount of fluorescing light that may be captured and quantified, thereby further reducing the measurement efficiency of a flow cytometry system. Another issue that further complicates flow cytometry measurements is that the fluorescing light that is ultimately delivered to a detector system capable of measuring the intensity of such fluorescing light may be extremely faint—so faint that many photodetector systems will be unable to adequately quantify it. To that end, extremely sensitive photodetector systems may be used, such as photomultiplier tubes, which convert the received florescent light into an electrical current that may be amplified by multiple orders of magnitude, e.g., 100,000 times.
In order to allow for a single flow cytometer to be used to process multiple different types of particles or cells and indicators, either separately or concurrently, many flow cytometers may include multiple photodetector systems, each equipped with a filtering system that allows for the flow cytometer to be easily reconfigured by removing or exchanging the filters. This allows each photodetector to be tuned to be receptive to only a particular spectrum of light, thereby allowing each photodetector to be used to detect the presence of a different indicator (or the presence of a different spectrum of received light—in some cases, an indicator may emit multiple different frequencies of light, and multiple different photodetectors may be used to detect each separate frequency).
Discussed herein are techniques and systems that improve upon flow cytometer systems having such reconfigurable filtering systems.